The Human Immunodeficiency Virus (HIV) infects cells by fusing its envelope membrane with the cell membrane through a process that is mediated by the viral envelope (Env) glycoprotein. During the fusion reaction, Env undergoes extensive conformational changes that are triggered upon sequential interactions with CD4 and coreceptors. These conformational changes are associated with transient exposure of conserved functionally important domains that are normally inaccessible to antibodies. In spite of this exposure, neutralizing antibodies against these domains are rare. The determinants of the ability of antibodies to neutralize HIV are poorly understood. In addition to steric factors that appear to limit the access to critical Env epitopes there is evidence that antibody binding to CD4-induced (CD4i) epitopes is kinetically restricted by brief exposure of these domains. We have recently shown that HIV-1 is unable to fuse with the plasma membrane and enters cells via dynamin-dependent endocytosis. Endosomal fusion occurs after a long delay following the virus uptake, suggesting that the relatively quick HIV-1 internalization could protect the virus from antibodies and fusion inhibitors targeting Env intermediates. We hypothesize that the efficacy of CD4i antibodies is determined in part by the time of exposure of proper Env epitopes on the cell surface. To test this hypothesis, we will increase the lifetime of Env intermediates and determine whether this intervention sensitizes the HIV-1 to neutralizing antibodies. To establish the relevance of this approach to enhancing the activity of CD4i antibodies in vivo, we will first verify that HIV-1 enters primary CD4+ T cells via endocytosis (Aim 1). The knowledge of the preferred HIV-1 entry route into these cells will guide our efforts to enhance the efficacy of CD4i antibodies. Specifically, the lifetime of the CD4-bound state will be extended by lowering the density of coreceptors (Aim 2a), whereas the residence time in the CD4/coreceptor-bound state will be prolonged by inhibiting productive HIV-1 endocytosis (Aim 2b). Through the newly developed kinetic assays that permit the time-resolved measurement of the HIV-1 progression through CD4- and CD4/coreceptor-bound states on the cell surface, the lifetimes of these key intermediates will be determined and correlated with the ability of CD4i antibodies to block fusion. These studies are expected to elucidate the role of kinetic factors in HIV-1 neutralization and to validate novel approaches aimed at enhancing the activity of CD4i antibodies. PUBLIC HEALTH RELEVANCE: The Human Immunodeficiency Virus (HIV) infects cells by fusing its envelope with the host cell membrane through a process that is mediated by the viral envelope (Env) glycoprotein. In spite of the transient exposure of conserved Env regions during HIV fusion, neutralizing antibodies recognizing these regions are rare. To test the hypothesis that antibody binding to conserved Env regions is restricted due to their brief exposure, we will stabilize intermediate conformations of Env on the cell surface and determine whether this intervention enhances the neutralizing activity of antibodies against these regions.